Carbohydrates play a number of important roles in the functioning of living organisms. In addition to their metabolic roles, carbohydrates are structural components of the human body covalently attached to numerous other entities such as proteins and lipids (called glycoconjugates). For example, human bone consists of hydroxyapatite, minerals, collagen protein, and a proteoglycan matrix. The carbohydrate portion of this proteoglycan matrix imparts essential properties to the bone structure and plays a role in bone metabolism.
Bone resorption is an important process in human health. Children have extremely high bone resorption rates due to their overall high rate of bone metabolism associated with growth. Bettica et al. (1992), Clinical Chemistry 38:2313-2518; Hanson et al. (1992), J. Bone Mineral Res. 7:1251-58. However, in adults, high rates of bone resorption are generally associated with metabolic bone disorders, such as primary hyperparathyroidism, osteomalacia, and Paget's disease. Seyedin et al. (1993), J. Bone Mineral Res. 8:635-41; Hanson et al. (1992), supra. Paget's disease, in particular, is characterized by a very high of bone resorption. Bettica et al. (1992), supra; Seyedin et al. (1993), supra. Although these diseases are significant, they affect a relatively small proportion of the population. The most widespread disease that is associated with a high rate of bone resorption in adults is osteoporosis; however, the increase in bone resorption associated with osteoporosis is much lower than that of Paget's disease, and hence is much more difficult to detect using conventional methods.
Osteoporosis is clinically characterized by low bone mass and abnormalities in the microarchitecture of the bone tissue, which lead to a reduction in skeletal strength and an increased susceptibility to fractures. WHO Technical Report #843 (1994). This disease reportedly affects between 15-20 million Americans (Mundy (1995), in Bone Remodeling and Its Disorders (Martin Dunitz Ltd., London), Chap. 12, p. 173), but these numbers may be an underestimate due to the lack of early diagnostic techniques. During the initial stages of this disease, clinical symptoms are subtle and not recognized by the patient. By the time clinical symptoms manifest, a significant amount of bone mass has already been lost. It has been estimated that nearly 45% of American women over the age of 50 have significant reductions in skeletal bone mass, putting them at an increased risk of vertebral, hip, or distal forearm fractures. Melton et al. (1992), J. Bone Mineral Res. 7:1005-10. More than 1.5 million Americans suffer from osteoporotic fractures every year, at an estimated cost to the health care industry of nearly $10 billion. Riggs and Melton (1992), New. Eng. J. Med. 327:620-27; Khosla and Riggs (1995), Mayo Clin. Proc. 70:978-82.
Preventive techniques to control the onset of osteoporosis include estrogen replacement therapy (Bonde et al. (1995), J. Clin. Endocrinol. Metab. 80:864-68; Gambacciani et al. (1994), Obstetrics Gynecol. 83:392-96) and calcium and vitamin D supplementation (Consensus development conference: Diagnosis, prophylaxis, and treatment of osteoporosis (1993), Am. J. Med. 94:646-50; Orimo et al. (1994), Calcified Tissue Int'l 54:370-76). Therapeutic interventions include growth hormone (Kassem et al. (1994), J. Bone Mineral Res. 9:1365-70), fluoride (Khosla and Riggs (1995), supra; Consensus development conference (1993), supra), bisphosphonates (Chestnut et al. (1995), Am. J. Med. 99:144-52; Reid et al. (1994), J. Clin. Endocrinol. Metab. 79:1595-99) and calcitonin (Khosla and Riggs (1995), supra).
These prevention and intervention therapies require accurate and sensitive measures of bone formation and bone resorption to be able to adequately assess their immediate and long-term efficacy. One of the best measures of susceptibility to fractures is assessment of bone mass or bone mineral density. WHO Technical Report #843 (1994). Although this method of monitoring bone mineral density yields measurements which have been shown to be highly correlated with bone strength, they do not provide information on the dynamics of bone resorption versus bone formation. Radiological techniques also provide information only about selected skeletal sites, complicated by the fact that each site has a different ratio of trabecular to cortical bone, which in turn have different rates of loss. Mundy (1995), supra. Most importantly, radiological techniques are relatively insensitive to changes in bone density which occur over time periods of less than one year. Measuring changes in bone density at intervals of more than one year, however, can result in significant bone loss before the disease is detected and treatment initiated. This measurement time lag also creates difficulties in assessing the efficacy of treatment interventions. It is clear that more accurate and sensitive methods for early detection of osteopenia and the subsequent risk for development of osteoporosis are needed.
A number of new biochemical markers for bone have been proposed for estimating rates of bone resorption, with the hope of allowing for early prediction of bone loss rather than later observation of reduced bone mineral density. Biochemical tests for bone resorption which are currently being tested include urinary hydroxyproline (Reeve et al. (1995), Calcified Tissue Int'l 57:105-10), serum cross-linked N- and C-telopeptides of type I collagen (Gertz et al. (1994), J. Bone Mineral Res. 9:135-42; Valimaki et al. (1994), Eur. J. Endocrinol 131:258-62), and urinary pyridinium cross-links (Valimaki et al. (1994), supra). These tests have become the most commonly discussed methods for detecting bone resorption and osteoporosis. For example, reviews of the currently known methods for detecting osteoporosis may be found in Valimaki et al. (1994), supra; Reeve et al. (1994), supra; Garnero et al. (1994), J. Clin. Endocrinol. Metab. 79:1693-1700; McCarroll (1993), Analytical Chem. 65:388R-95R (review article); Bettica et al. (1992), supra. However, these tests have high within-subject variability. Furthermore, most urinary biochemical markers are normalized to creatinine to account for daily fluctuations in urine concentration. This normalization creates several problems, such as particularly high variation between samples collected at different times during the day (sometimes as high as 30%). This problem is most serious if excretion rates of abnormals and normals are fairly close, because a high coefficient of variation between samples would tend to produce higher numbers of false negatives.
In view of the problems of these markers, diagnosis would be helped greatly if more sensitive and convenient techniques could be developed for the screening and diagnosis of osteoporosis. In assessing these new tests, it is important to establish that they can, in practice, be used to monitor the development of osteoporosis. Falch (1994), Scand. J. Clin. Lab. Invest. (Suppl. 219) 54:40-41; Farley and Baylink (1995), Clin. Chem. 41:1551-53. The validation of these new urine and serum assays is perhaps even more urgent due to the need for immediate clinical assessment of emerging new treatments of osteoporosis. Garnero et al. (1994), supra. Ideally, these tests should be noninvasive, convenient, accurate, sensitive, specific to the disease process, and economical.
Several authors have speculated that glycosaminoglycans (GAGS) as a broad chemical class could be evaluated as biochemical markers for bone turnover. Larking et al. (1987), Biochem. Med. Metabol. Biol. 37:246-54; Todorova et al. (1992), Horm. Metab. Res. 24:585-87; McCarroll (1993), supra. GAGs are carbohydrates which are integrally related to collagen and comprise a portion of proteoglycans found in connective tissue and bone. Mundy (1995), supra. Specifically, GAGs are sugar chains consisting of repeating polymers of acidic polysaccharides. These materials are composed of building blocks of the following sugars in various combinations: galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, galacturonic acid and iduronic acid. In addition, these sugar units may be variably linked .alpha. or .beta. at their anomeric carbons and (1-3) or (1-4) to their ring carbons through an O-glycosidic bond. Finally they may be variably substituted with sulfates at their 2,3,4 or 6 carbons. Depending on the precise repeating disaccharide structure and location of sulfates, human connective tissue GAGs are commonly classified as chondroitin sulfates, dermatan sulfates, heparan sulfates, heparin sulfates and keratan sulfates. Collins (1987), Carbohydrates (Chapman Hall, London). Degradation products of these GAGs are found in human urine and serum, with chondroitin sulfate being the major GAG of normal urine (Poulsen (1981), Scand. J. Clin. Lab. Invest. 41:675-81).
One method of measuring individual carbohydrates in a sample is by FACE, an acronym standing for the technique of Fluorophore-Assisted Carbohydrate Electrophoresis. The FACE technique is described in detail in U.S. Pat. Nos. 4,975,165, 5,035,786, 5,104,508, 5,109,231, 5,205,917, 5,316,638, 5,340,453, 5,472,582, and 5,087,337. However, until the present invention, no one has been able to use FACE (or for that matter, any other technique) to assay accurately the levels of specific GAGs in urine. Additionally, those skilled in the art believed other biochemical markers were more suitable than GAGS for assaying bone resorption, and so most references teach the use of these other markers. Indeed, in 5 articles comparing biochemical markers of bone resorption (Valimaki et al. (1994), supra; Reeve et al. (1994), supra; Garnero et al. (1994), supra; McCarroll (1993), supra; Bettica et al. (1992), supra), and only one mentions GAGs (McCarroll (1993), supra) and only in passing. Moreover, no one, until the present invention, even discusses chondroitin sulfate specifically as a potential biochemical marker.